The innate immunity is comprised of several types of cells including dendritic cells (DC's), macrophages and monocytes, polymorphonuclear cells, natural killer (NK) cells, innate lymphoid cells and natural killer T cells (NKT cells) which detects various pathogens as well as aberrant host cells with potential for danger to tissue integrity through specialized receptors like toll-like receptors. Toll-like receptors (TLRs) are a family of germline-encoded cell surface pattern recognition molecules containing an pathogen binding ectodomain (ECD) with 19-25 leucine-rich repeats (LRRs), a transmembrane domain and a characteristic cytoplasmic domain called the TIR (Toll/IL-1 receptor) domain. TIR domain is responsible for downstream signalling, whereas LRRs containing 24-29 amino acids are responsible for ligand recognition and binding. TLRs get triggered in response to bacterial and fungal infections (Medzhitov, R; Nat. Rev. Immunol. 1, 135-145, 2001) followed by induction of downstream signalling, leading to expression of inflammatory genes like those of the nuclear factor-κB (NF-κB) family of transcription factors and antimicrobial peptides. There are 11 human and 12 mice TLRs have been identified which recognize different molecular patterns on the pathogens.
Major group of the TLRs are expressed on the cell surface. The leucine-rich repeats in the ectodomains of these molecules bind to unique molecular entities on pathogens (PAMPs), which detect and initiate responses to invading microorganisms (Akira, S; et al. Annu Rev Immunol. 21, 335-76, 2003). Another group of TLRs (endosomal TLRs) are located inside the cell within the endosomal-lysosomal compartments, instead of being expressed on the cell surface (Akira, S; et al. Annu Rev Immunol. 21, 335-76, 2003). This group comprises of TLR3 (Alexopoulou, L; et al. Nature, 413(6857), 732-8, 2001), TLR7 (Hemmi, H; et al. Nature, 408(6813), 740-5, 2001; Lund, J. M; et al. Proc Natl Acad Sci USA. 101(15), 5598-603, 2004), TLR8 (Heil, F; et al. Science, 303(5663), 1526-9, 2004) and TLR9 (Hemmi, H; et al. Nature, 408(6813), 740-5, 2001). The endosomal TLRs are specialized for detecting microbial nucleic acids after microbes get phagocytosed and reach the endosomal compartments.
The downstream signalling goes through recruitment of intracellular adaptor molecules such as Myd88 (or the myeloid differentiation primary-response gene 88), TIRAP (or the TIR-domain containing adaptor protein), TRIF (or the TIRAP inducing IFN-beta) and TRAM (or the TRIF-related adaptor molecule). TLR-adaptor molecule interactions in turn recruit other proteins to the signalling complex, which initiates multiple downstream signalling pathways, leading to activation of NFkB or mitogen-activated protein kinases (MAPKs) or recruitment of the IFN regulatory factors (IRFs). These different pathways in turn result in the transcription of genes encoding different cytokines, chemokines, co-stimulatory molecules or other proteins, thereby sculpting the ensuing immune response (Akira, S; et al. Annu Rev Immunol. 21, 335-76, 2003). The intracellular localization of the nucleic acid-recognizing TLRs (TLR3, 7, 8, 9) is one of the mechanisms that prevent their spontaneous activation by circulating host-derived nucleic acids (Barton, G. M; et al. Nat Immunol. 7(1):49-56, 2006), however under certain pathological conditions the endogenous nucleic acids can overcome this regulation. It has been previously shown by us and others that the circulating immune complexes found in sera of patients suffering from systemic lupus erythematosus (SLE) typically contain nucleic acids associated with various proteins such as antibodies, the chromatin-associated protein HMGB1, the antimicrobial peptide LL37, ribonuclear proteins and others (Lande, R; et al. Nature, 449(7162), 564-9, 2011; Ganguly, D. et al. Nat Rev Immunol. 13(8), 566-77, 2013). Our previous studies have also shown that TLR9, 7 and 8 activation driven by self nucleic acid and LL37 complexes may also play an important pathogenic role in Psoriasis (Lande, R; et al. Nature, 449(7162), 564-9, 2007; Ganguly, D. et al. J Exp Med. 206(9), 1983-94, 2009). These associated proteins may protect the bound nucleic acid from degradation and/or facilitate their entry into the cell, as is the case for Fc receptor-mediated uptake of antibody-nucleic acid complexes (Leadbetter, F. A; et al. Nature, 416(6881), 603-7, 2002; Ganguly, D. et al. J Exp Med. 206(9), 1983-94, 2009). Once inside the endolysosomal compartments, the nucleic acid cargo can then stimulate the intracellular TLRs, priming the immune system for a cascade of inflammation inciting cytotoxic and/or humoral response. For example, this cycle of innate immune recognition, generation of autoreactive antibodies, and consequent immune complex formation is believed to play critical role in the pathogenesis of SLE and possibly Sjogren's syndrome (Marshak-Rothstein, A; Nat Rev Immunol. 6(11), 823-35, 2006; Lande, R; et al. Nature, 449(7162), 564-9, 2011; Ganguly, D. et al. Nat Rev Immunol. 13(8), 566-77, 2013), a finding confirmed in animal models treated with TLR7 and TLR9-competitive antagonist oligonucleotides (Banat, F. J; et al. Eur J Immunol. 37(12), 3582-6, 2007; Christensen, S. R; et al. J Exp Med. 202(2), 321-31, 2005). TLR-mediated pathological responses to nucleic acids have also been shown to contribute to other pathologies like psoriasis (Lande R et al, Nature, 2007; Ganguly D et al, J Exp Med, 2009), ischemic liver injury (Bamboat, Z. M; et al. Hepatology, 51(2), 621-32, 2010) lung infection (Itagaki, K; et al. Shock, 36(6), 548-52, 2011), pancreatitis (Hogue, R; et al. Gastroenterology, 141(1), 358-69, 2011) and graft-versus-host disease (Calcaterra, C; et al. J Immunol. 181(9), 6132-9, 2008).
Hydroxychloroquine and chloroquine are not only used as anti-malarial agents but has been commonly prescribed to treat various clinical contexts of autoreactive inflammation (autoimmune diseases) such as rheumatoid arthritis (RA) and SLE (Wallace, D. J; Lupus, 5 Suppl 1, S59-64, 1996). In literature there are several reports of small molecule analogues and derivatives of chloroquine with substituted quinoline and quinazoline scaffold which can inhibit stimulation of the immune system. U.S. Pat. Nos. 6,221,882; 6,479,504; 7,410,975 B2; WO 2008/030455, published Mar. 13, 2008; U.S. Pat. No. 7,410,975 B2; PCT published application PCT/US03/17733 (WO 03/103586 A2); and PCT published application PCT/US2009/058401 (WO2010/036908).